An understanding of the regulation of protein metabolism in skeletal muscle is required for the development of rational approaches to the treatment of diabetes mellitus and other disease states that affect nutrient homeostasis. Toward this end, the overall goal of this project is to gain a better understanding of the mechanisms through which insulin acts to regulate protein synthesis in skeletal muscle. The project proposes to build upon major advances achieved during the past grant period in regards to our understanding of the initiation of mRNA translation and the signaling mechanisms linking the control of translation to cell surface insulin receptors. Translation initiation requires a number of specific proteins termed initiation factors (in eukaryotes, eIF's). It has two main control points, each an important target of insulin action: binding the initiator methionyl-tRNA to the 40S ribosomal subunit, mediated by eIF-2 and regulated by eIF-2B; and attachment of mRNA to the 40S ribosomal subunit, mediated by eIF-4F (a complex comprised of elF-4A, eIF-4E, and eIF-4Fgamma) and regulated in part by the eIF-4E binding protein termed PHAS-I or 4E-BP1. Insulin action appears to be mediated by changes in phosphorylation of the epsilon-subunit of eIF-2B, PHAS-I, and components of the eIF-4F complex. The specific aims of the project described here are: 1) to identify the serine and/or threonine residues in eIF-2Bepsilon, PHAS-1, and the proteins comprising the eIF-4F complex that undergo changes in phosphorylation in response to insulin 2) to identify and characterize the protein kinases involved in the insulin-mediated changes in phosphorylation of the serine and/or threonine residues identified above; 3) to investigate the signal transduction pathways utilized to control the insulin regulated kinases identified above by employing specific inhibitors and/or by identifying and characterizing the relevant kinase kinases; 4) to investigate the mechanism whereby changes in phosphorylation of eIF-2B alters its function; and 5) to investigate the mechanism whereby changes in phosphorylation of PHAS-I and of the proteins comprising the eIF-4F complex alters the binding of mRNA to the 40S ribosomal subunit. Overall, the studies described here should help to identify mechanisms through which insulin acts to regulate protein synthesis in skeletal muscle. They should also provide new insights into the biochemical and molecular mechanisms involved in translation initiation.